Integrated microbial collector

ABSTRACT

A system for real-time sizing of fluid-borne particles is disclosed. The system further determines, in real time, whether the detected particles are biological or non-biological. As the fluid is being tested, it is exposed to a microbe collection filter which is cultured to determine the type of microbes present in the fluid being tested.

CROSS-REFERENCE TO RELATED APPLICATION

This is a Continuation Application which claims priority from U.S.Non-Provisional application having Ser. No. 13/739,730, filed on Jan.11, 2013, which is a Continuation Application which claims priority fromU.S. Non-Provisional application having Ser. No. 12/642,722, filed onDec. 18, 2009, to issue as U.S. Pat. No. 8,358,411, which in turn claimspriority to U.S. Provisional Application having Ser. No. 61/138,878,filed Dec. 18, 2008. The contents of each of these applications areentirely incorporated herein by reference in their entirety, for allpurposes.

FIELD OF INVENTION

The present invention relates to generally to a system and method fordetecting airborne or liquid-borne particles (generally, fluid-borneparticles), and more particularly to a system and method for detectingairborne or liquid-borne particles, determining the size of theparticles, determining the particles' status as biological or inert, andclassifying the type of biologic particle detected.

BACKGROUND OF INVENTION

A variety of manufacturing environments require strict control over thepresence of foreign debris in the air. Semiconductor manufacturing, forexample, has long required “clean-rooms” that use extensive airfiltering to reduce the number and size of particles in the air to someacceptable level. Other manufacturing environments have similar butdistinct requirements. For example, in pharmaceutical or medical devicemanufacturing environments it is critical to control not only the numberof particles in the air, but minimization of biologic particles is ofparticular importance. Microbial contamination, for example, can renderan entire batch of pharmaceutical product unusable leading tosignificant monetary losses in the manufacturing process. Additionally,it is advantageous to have instantaneous detection of contaminationevents, including instantaneous information about whether acontamination event is biologic or non-biologic, during themanufacturing process for pharmaceuticals or medical devices.

A variety of systems and methods exist that provide instantaneousdetection of fluid borne particles. For example, certain detectors havebeen designed to detect fluid borne particles and provide warning whenthe number of particles within an air sample exceeds a predeterminedminimum value. Exemplary devices are described in U.S. Pat. Nos.5,646,597, 5,969,622, 5,986,555, 6,008,729, and 6,087,947, all toHamburger et al. These detectors all involve direction of a light beamthrough a sample of environmental air such that part of the beam will bescattered by any particles in the air, a beam blocking device fortransmitting only light scattered in a predetermined angular rangecorresponding to the predetermined allergen size range, and a detectorfor detecting the transmittal light. An alarm is actuated if the lightdetected at the detector is above a predetermined level.

Additionally, systems and methods exist that instantaneously determinewhether detected particles are biologic or inert. For example, U.S. Pat.No. 7,430,046 to Jiang et al., discloses systems and methods forsimultaneously measuring particle size by use of Mie scattering anddetermining whether the measured particles are biologic or inert bydetecting fluorescence excited in certain biological chemicals presentin the measured particles.

Although fluorescence analysis can, in certain cases, be used todetermine the type of biologic particle detected, i.e., the type oforganism, it would be advantageous to have additional systems andmethods that could simultaneously collect information on particle size,whether a particle is biologic or non-biologic, and the type of biologicparticle that has been detected.

SUMMARY OF THE INVENTION

Embodiments of the invention continuously sample fluid containingparticles from an environment to be monitored. Sampled fluid is passedthrough a sampling area, where it is exposed to light. Light scatteredby the particles in the fluid is detected and used to determine the sizeof the particles. Light emitted by fluorescence from the particles isalso detected and used to determine whether the particles are biologicalor non-biological. A filter containing a gas-permeable substance towhich microbes adhere, (i.e., a microbe collection filter) is placedafter the sampling area, such that the microbe collection filter isplaced into the flow of the fluid being measured. In certainembodiments, the filter is situated atop a circular support plateincluding perforations allowing fluid to flow through the plate. Themicrobe collection filter collects biological particles from at least aportion of the flow of fluid. After optical measurements have beenperformed for a predetermined amount of time, and microbe collectionfilter has been exposed, the filter is removed, microbial nutrientsand/or water are added if necessary, and the filter is incubated andexamined for the growth of organisms according to any of a number ofmeans, e.g., colony counting, observing the macroscopic appearance ofthe growth patterns of microbes on the filter, microscope observation ofthe microbes or chemical testing for the metabolic by-products ofmicrobial growth.

In one embodiment, a microbial detection and identification system isdescribed. The system includes a sampling area including a fluid, alight source, a first optical detector and a second optical detector.The system further includes a microbe collection filter in fluidcommunication with the sampling area. The light source illuminatesparticles in the sampling area, the first detector detects lightscattered into a predetermined angular range by particles of apredetermined size, and the second detector measures light emitted byfluorescence from illuminated biological particles in the sample area.Additionally, the fluid is exposed to the microbe collection filtercausing particles in the fluid to adhere to the microbe collectionfilter.

In certain embodiments, a first optical system directs scattered lightfrom the sampling area to the first detector and a second optical systemthat directs fluorescence light from the sampling area to the seconddetector. In certain embodiments, the second optical system includes along-pass filter that selectively transmits light having a wavelength oflight emitted by fluorescence by illuminated particles in the samplearea. In certain embodiments, an ellipsoidal reflector is includedhaving a first focus at the sampling area and a second focus near thesecond detector.

In certain embodiments, the long-pass optical filter comprises twolong-pass reflective filters in series. In certain embodiments, thelight source comprises an LED or a diode laser, and the light sourceemits at a wavelength of approximately between 350 nm and 410 nm. Incertain embodiments, the microbe collection filter includes a firstsurface, a second surface, and a gas permeable interior allowing thefluid to flow into the first surface, and out of the second surface. Incertain embodiments, the microbe collection filter is gelatin.

In some embodiments, the fluid is air, and additional embodimentsinclude a blower in fluid communication with the sampling area, wherethe blower supplies negative pressure to the sampling area therebydrawing environmental air into the sampling area and evacuating air fromthe sampling area as it is optically measured. In certain embodiments,the blower is in fluid communication with the microbe collection filter,and air is drawn through the microbe collection filter after it isoptically measured.

Certain embodiments recite a method of detecting microbial contaminationin a fluid. The method involves illuminating a fluid with a lightsource, detecting particles of a pre-determined size range present inthe fluid by measuring light scattered by illuminated particles into apredetermined range of angles, classifying particles in the fluid asbiological or non-biological by measuring fluorescent light emitted fromilluminated particles, and exposing a microbe collection filter to thefluid.

Certain embodiments include storing data related to the detection ofparticles of a predetermined size range and classification of particlesas biological or non-biological for further analysis. Additionalembodiments include culturing the exposed microbe collection filter.Other embodiments include analyzing the cultured exposed microbecollection filter to determine the types of microbes present on themicrobe collection filter at the time of exposure.

Certain embodiments include analyzing the cultured exposed microbecollection filter comprises one or more of: colony counting, observingthe macroscopic appearance of the growth patterns of microbes on thecultured exposed microbe collection filter, microscope observation ofmicrobes on cultured exposed microbe collection filter, and chemicaltesting for the metabolic by-products of microbial growth present on thecultured exposed microbe collection filter. Other embodiments includestoring data related to the detection of particles of a predeterminedsize range and classification of particles as biological ornon-biological for further analysis. Certain embodiments includecorrelating the determination of the types of microbes present on themicrobe collection filter at the time of exposure with stored datarelated to size of particles present in the fluid at the time ofexposure and the status of the particles as biological ornon-biological.

Certain embodiments include a method of characterizing a contaminationevent. The method involves illuminating a first fluid with a lightsource, detecting particles of a pre-determined size range present inthe first fluid by measuring light scattered by illuminated particlesinto a predetermined range of angles, and classifying particles in thefirst fluid as biological or non-biological by measuring fluorescentlight emitted from illuminated particles. The method also includes,storing data related to a scattering and fluorescence characteristics ofparticles in the first fluid, exposing a microbe collection filter tothe first fluid, culturing the microbe collection filter and analyzingthe cultured exposed microbe collection filter to determine the types ofmicrobes present on the microbe collection filter at the time ofexposure. The method also includes correlating the determination of thetypes of microbes present on the microbe collection filter at the timeof exposure with stored data related to the scattering and fluorescencecharacteristics of particles in the first fluid, optically detectingscattering and fluorescence characteristics of particles in a secondfluid, and comparing the detected scattering and fluorescencecharacteristics in the second fluid with the stored data related to thescattering and fluorescence characteristics of particles in the firstfluid. Other embodiments are directed to a method where on the basis ofthe comparison, determining that microbes present in the first fluid arelikely present in the second fluid.

Advantages of the invention include the ability to performinstantaneous, simultaneous particle sizing and detection of biologicalor non-biological organisms. Additional advantages include the abilityto determine the type of biological particle detected and correlate dataon the type of organism detected with the real-time particle data tofully characterize a contamination event. Additional advantages includethe possibility of predicting the type of microbial contaminationassociated with future events on the basis of past correlations betweenoptical and growth-medium measurements of particles.

BRIEF DESCRIPTION OF THE DRAWINGS

Implementations of the invention will become more apparent from thedetailed description set forth below when taken in conjunction with thedrawings, in which like elements bear like reference numerals.

FIG. 1 is a schematic diagram of a system for instantaneous andsimultaneous particle sizing and biological detection according to anembodiment of the invention.

FIG. 2 is a schematic diagram showing the microbial collector componentsof a particle sizing and biological detection system according to anembodiment of the invention in additional detail.

FIG. 3 is an exploded drawing showing an assembly for holding a microbecollection filter according to an embodiment of the invention.

FIG. 4 is a plan view of an exemplary filter support plate suitable foruse in conjunction with an embodiment of the invention.

FIG. 5 is a schematic flowchart illustrating steps of a method ofmicrobial detection and analysis according to an embodiment of theinvention.

FIG. 6 is a schematic block diagram of a computer-automated machinevision and analysis system according to an embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

This invention is described in preferred embodiments in the followingdescription with reference to the Figures, in which like numbersrepresent the same or similar elements. Reference throughout thisspecification to “one embodiment,” “an embodiment,” or similar languagemeans that a particular feature, structure, or characteristic describedin connection with the embodiment is included in at least one embodimentof the present invention. Thus, appearances of the phrases “in oneembodiment,” “in an embodiment,” and similar language throughout thisspecification may, but do not necessarily, all refer to the sameembodiment.

The described features, structures, or characteristics of the inventionmay be combined in any suitable manner in one or more embodiments. Inthe following description, numerous specific details are recited toprovide a thorough understanding of embodiments of the invention. Oneskilled in the relevant art will recognize, however, that the inventionmay be practiced without one or more of the specific details, or withother methods, components, materials, and so forth. In other instances,well-known structures, materials, or operations are not shown ordescribed in detail to avoid obscuring aspects of the invention.

The schematic flow charts included are generally set forth as logicalflow chart diagrams. As such, the depicted order and labeled steps areindicative of one embodiment of the presented method. Other steps andmethods may be conceived that are equivalent in function, logic, oreffect to one or more steps, or portions thereof, of the illustratedmethod. Additionally, the format and symbols employed are provided toexplain the logical steps of the method and are understood not to limitthe scope of the method. Although various arrow types and line types maybe employed in the flow chart diagrams, they are understood not to limitthe scope of the corresponding method. Indeed, some arrows or otherconnectors may be used to indicate only the logical flow of the method.For instance, an arrow may indicate a waiting or monitoring period ofunspecified duration between enumerated steps of the depicted method.Additionally, the order in which a particular method occurs may or maynot strictly adhere to the order of the corresponding steps shown.

FIG. 1 shows a system for instantaneous particle sizing and biologicalparticle detection according to an embodiment of the invention. Thesystem of FIG. 1 includes a housing 102, which serves to seal and/orisolate the interior components of the system from contamination fromthe surrounding air and interference from light sources in theenvironment of the system.

The system of FIG. 1 further includes a light source 105. In oneembodiment, light source 105 produces an output having a wavelengthbetween 270 nm and 410 nm. In one embodiment, light source 105 producesan output having a wavelength between 350 nm and 410 nm. In oneembodiment, light source 105 produces an output having a wavelength ofabout 405 nm. The spectral characteristics of light source 105 are suchthat light emitted by light source 105 is capable of undergoing Miescattering when interacting with particles of a size range of interest.Additionally, light source 105 is selected to have a wavelength capableof exciting intrinsic fluorescence from metabolites inside microbes andother biological particles. A wavelength of about 270 nm to 410 nm ischosen based on the observation that microbes and biological particlescontain at least one of a number of primary metabolites that exhibitfluorescence: tryptophan, which normally fluoresces at excitationwavelengths of about 270 nm, with a range of about 220 nm to about 300nm; nicotinamide adenine dinucleoetide (NADH), which normally fluorescesat excitation wavelengths of about 240 nm, with a range of about 320 nmto about 420 nm; and riboflavin, which normally fluoresces atwavelengths of about 200 nm, with a range of between 320 nm to about 420nm. In the case of bacterial endospores, dipicolinic acid (DPA) normallyfluoresces at excitation wavelengths of about 400 nm, with a range ofabout 320 nm to about 420 nm. A light source having a wavelength outputof about 350 nm to about 410 nm ensures excitation of two of the threeaforesaid primary metabolites: NADH and riboflavin, as well as DPA.Selection of this wavelength band allows for fluorescence to begenerated and detected in bio-particles, but excludes excitation ofnon-biological sources of florescence such as diesel engine exhaust andother inert particles such as dust or baby powder.

Light source 105 can be a laser such as a diode laser, an LED or aspectrally filtered broadband source such as a lamp. Light source 105can optionally include collimating or beam shaping optics to produce asubstantially collimated output and/or an output having a transversebeam profile that is flat, in terms of power. Optionally, light source105 includes an optical fiber that delivers light to the vicinity of theother elements of the system of FIG. 1. When an optical fiber is used todeliver light from a remotely situated light source, collimating or beamshaping optics may optionally be provided at the output of the opticalfiber.

Light source 105 provides a substantially collimated beam of light to atleast a portion of the sampling area 110. The intersection of thesubstantially collimated beam from light source 110 and the samplingarea creates an interrogation zone, which is an illuminated portion ofthe sampling area. In one embodiment, where the fluid to be measured isair or some other gas, sampling area 110 is defined by the space betweentwo air nozzles that provide air flow through the sampling area. In theembodiment of FIG. 1, sampling area 110 is defined by the space betweentwo nozzles, an entrance nozzle 112, which supplies air to the samplingarea, and an exit nozzle 114, which extracts air from the sampling area.

Upon illumination from light source 105, particles within sampling area110 scatter light by Mie scattering. Mie scattering generally scatterslight at angles inversely proportional to particle size. Accordingly,relatively small particles will scatter light at higher angles relativeto the scattering produced by relatively larger particles. In practice,scattered light emerges from sampling area 110 in a cone centered aboutan axis defined by the collimated beam emerging from light source 105.The amount of light scattered into various angles is used, according tocertain embodiments of the invention, to determine the size of theparticles scattering light.

The system of FIG. 1 further includes a scattered light collection lens115. In one embodiment, scattered light collection lens is aplano-convex lens arranged with the plano side facing toward samplingarea 110 to minimize the spherical aberration associated with collectingand collimating scattered light. Scattered light collection lens 115collects and collimates light scattered at relatively high angles byparticles in sampling area 110 by being configured and positioned suchthat its front focal plane is co-incident with sampling area 110.

The system of FIG. 1 further includes a scattered light condenser lens120. Scattered light condenser lens 120 takes collimated light emergingfrom scattered light collection lens 115 and focuses that light ontoscattered light detector 125, which generates an electrical signal inproportion to the amount of scattered light incident on detector 125. Inone embodiment, scattered light detector 125 is a photo-diode.

The system of FIG. 1 further includes beam blocking device 135. Beamblocking device 135 prevents further propagation of the collimated beamemitted by light source 105 after light emitted by light source 105 haspropagated through sampling area 110. In one embodiment, beam blockingdevice 135 is a disk of optically absorptive material of a diametersomewhat greater than the beam diameter of the collimated beam emittedby light source 105. In the embodiment of FIG. 1, beam blocking device135 is affixed to scattered light collection lens 115. In certainembodiments, beam blocking device 135 is a disc of black anodizedaluminum. In other embodiments, beam blocking device 135 is a smalllight box with an absorptive interior coating arranged to force lightemitted by light source 105 and entering the light box to undergomultiple internal reflections. Alternatively, beam blocking device is afold mirror that directs light emitted by light source 105 to a beamdump arranged at some position outside of the optical componentspictured. Alternatively, beam blocking device 135 is a conical shapewith an absorptive interior coating that the beam enters at the openside. Beam blocking device 135 can be any device or combination ofdevices that prevents further propagation of the collimated beam, orstray reflections caused by the collimated beam, emitted by light source105 after light emitted by light source 105 has propagated throughsampling area 110.

The scattered light detection components of the system of FIG. 1 arearranged such that only a specific range of angles of scattered lightare detected by scattered light detector 125. This can be accomplishedin a number of ways. For example, the scattered light collectioncomponents can be sized and/or positioned in such a way as to onlyintercept light that has been scattered into a predetermined angularrange of interest. Beam blocking device 135 inherently blocks very lowangle light, including the unscattered light that passes through thesampling area 110 from light source 105. In one embodiment, beamblocking device 135 is sized to block not only light that is propagatingalong an optical axis defined by the beam from light source 105, butalso light that is scattered at low, but still non-zero angles. In thisway, in certain embodiments, beam blocking device 135 is used to definea lower bound for the range of angles measured by the scattered lightmeasurement components.

In certain embodiments, an upper bound for the range of angles measuredby the scattered light measurement components is established by theposition and size of scattered light collection lens 115. Lightscattered at high angles will not be intercepted by scattered lightcollection lens 115, so the size and distance of scattered lightcollection lens from the plane of sampling area 110 defines an upperbound for the range of angles measured by the scattered lightmeasurement components. Additionally or alternatively, annular masks ofoptically non-transmissive material may be placed in the scattered lightdetection path, for example, on scattered light condenser lens 120 orthe scattered light collection lens 115 to limit the range of scatteringangles measured.

The system of FIG. 1 further includes an ellipsoidal reflector 130. Theshape of ellipsoidal reflector 130 is defined with respect to a vertexlocated off the axis defined by the beam of substantially collimatedlight emitted by light source 105. In other words, when viewed withrespect to the axis defined by light source 105, the collimated beamemitted by light source 105, sampling area 110 and the scattered lightcollection components, ellipsoidal reflector 130 is an off-axis ellipse.A first focus of ellipsoidal reflector 130 is located at the samplingarea 110 and is substantially co-incident with the front focal point ofscattered light collector lens 115. A second focus of ellipsoidalreflector 130 is located near the input port of a photomultiplier tube(“PMT”) 150, the function of which is set forth in further detail below.In one embodiment, ellipsoidal reflector 130 includes a circularaperture, for example, near its vertex, to allow for uninterruptedpropagation of light from light source 105 to sampling area 110.

Particles undergoing florescent emission within the sampling area 110will emit light isotropically, that is, will emit equal optical powerinto all angles defining a sphere. Ellipsoidal reflector 130 ispositioned such that it intercepts at least a portion of the lightemitted by fluorescence from particles within the sampling area. Thefluorescence light collected by parabolic reflector 130 at its firstfocus is directed along an axis defined by ellipsoidal reflector 130toward the second focus located near an input port to PMT 150.

The system of FIG. 1 further includes back-to-back long-pass opticalfilters 140, i.e., two long-pass, reflective interference filters inseries. In one embodiment, long-pass optical filters 140 are reflectiveinterference type filters that transmit light having a wavelength longerthan a certain wavelength while reflecting light having a wavelengthshorter than a certain wavelength. The spectral characteristics offilters 140 are such that light emitted by particles within the samplingarea 110 by fluorescence is transmitted, while light havingsubstantially the same wavelength as that emitted by light source 105 isreflected.

Since fluorescence results in the emission of light having a longerwavelength than the excitation wavelength, filters 140 pass only lightemitted by fluorescence, while reflecting noise (e.g., strayreflections) from the light source 105 as well as light scattered byparticles within the sampling area 110 at angles toward PMT 150. Twolong-pass filters are used in series to improve the performance of thefilters. This arrangement is advantageous when placing the filters 140in a converging beam, i.e., where the filters are used at non-zeroangles of incidence.

The system of FIG. 1 further includes blower 160. Blower 160 is arrangedto draw fluid in the sampling area out of the system after it has beenoptically interrogated.

It is important to note that the specifics of the optical collectionsystems for scattered and fluorescent light described above with respectto FIG. 1 are exemplary and not required. Any combination orconfiguration of optical system capable of simultaneously collecting andmeasuring scattered and fluorescent light should be deemed to be withinthe scope of embodiments of the invention.

FIG. 2 shows the microbial collection components of a system accordingto an embodiment of the invention. In certain embodiments, the microbialcollection components illustrated in FIG. 2 are used with the opticalsizing and biological detection components described in reference toFIG. 1. FIG. 2 omits the optical components for clarity, however. Thesystem of FIG. 2 includes a housing 202. In the system of FIG. 2, air tobe measured is routed to a sampling area 210 via an entrance nozzle 212.Once in sampling area 210, particles in the air are sized and classifiedas biological or non-biological by measuring the angular distribution ofscattered light and fluorescence. One exemplary way this is accomplishedby the operation of system components described above with respect toFIG. 1. After being measured optically in sampling area 210, air beingmeasured is extracted from sampling area by exit nozzle 214 where it ispassed to filter holder 260.

Filter holder 260 contains microbe collection filter 265. In oneembodiment, microbe collection filter 265 is a 47 mm gelatin plate thatcan be removed from filter holder 260 and placed in a conventional Petridish for incubation. Microbe collection filter 265 has a front sidefacing toward exit nozzle 214 and a rear side facing toward perforatedsupport plate 266, which itself includes perforations allowing fluidflowing through microbe collection filter 265 to flow the perforationsinto a rear chamber 267 of filter holder 260. Rear chamber 267 is influid communication with exit fluid line 269. Microbe collection filter265 is supported by perforated support plate 266, which includes aplurality of perforations allowing fluid to flow from the back side ofthe microbe collection filter 265 through the support plate 266. Filterholder 260 is configured to provide an air-tight seal, by the use ofcompressed o-rings or the like, such that substantially all of the airextracted from sampling area 210 through exit nozzle 214 flows throughthe microbe collection filter 265. From the back side of microbecollection filter 265, air flows through the perforations in supportplate 266, and through fluid exit line 269, which is in fluidcommunication with blower 270. Blower 270 draws fluid through exit line269, the perforations in support plate 266, and microbe collectionfilter 265. Since microbe collection filter 265 and its support plateprovide a partial obstruction to air flow, the suction created by blower270 at the front side of microbe collection filter 265 will be less thanthe suction created by blower 270 near the back side of microbecollection filter 265. The suction at the back side of microbecollection filter 265 is maintained at a level sufficient to extract theair being measured from sampling area 210. This is accomplished byselecting sufficiently high blower suction, and a sufficiently highcross-sectional perforation area of the gelatin filter's support plateto maintain adequate negative pressure at the exit nozzle 214.

In one embodiment, microbe collection filter 265 is a gas-permeablesubstance that causes microbial particles in fluid exposed to the filterto adhere to the filter. Additionally, microbe collection filter 265 isconfigured to maintain microbial viability so that microbes collected bythe filter can be cultured and analyzed. In particular embodiments,microbe collection filter 265 is a gelatin wafer having a sufficientlyhigh water content to maintain microbial viability, although the use ofgelatin is not a requirement. Any filter containing a gas permeablesubstance with a high moisture content to maintain biological viability,to which microbes present in fluid exposed to the filter adhere, isacceptable to use as the filter described herein.

In the embodiment of FIG. 2, microbial collection filter 265 isremovable so that, after a measurement period, the microbe collectionfilter 265 can be incubated and then analyzed to determine the number,and in certain embodiments, the types of microbes that were present inthe measured air during the measurement period. In an exemplary process,after exposure, microbial collection filter 265 is removed and coveredwith a sterile cover for transport. After transport, microbialcollection filter 265 is placed in contact with a growth mediumcontaining plate, e.g., an agar containing Petri dish. The filter isthen incubated for some amount of time, and the resulting microbialgrowth is analyzed. Analysis can take several forms. For example,microbial colonies may be visually visible and can be counted todetermine the number of microbes that landed on the microbial collectionfilter during the measurement period. Additional analyses may beperformed, for example, in certain cases the shape, i.e., themacroscopic appearance, of a microbial colony can provide informationabout the type of microbe present. Additionally or alternatively,microscopic observation of the microbes, chemical testing for themetabolic by-products of microbial growth and/or DNA analysis candetermine the type of microbe collected.

FIG. 3 is an exploded drawing showing a microbe collection filterassembly according to an embodiment of the invention. FIG. 3 shows apreferred embodiment for the filter holder 260 described above withrespect to FIG. 2. Microbe collection filter assembly includes cartridgehousing 305. Cartridge housing 305 includes hose barb 310 and flowsensor tube 315 which are arranged at sensor input aperture 320 one theside of the cartridge housing. This sensor assembly can be used tomonitor air flow by the Venturi effect or the like. Fluid exiting thesystem enters cartridge housing, and therefore filter assembly, viainput aperture 321 which is in fluid communication with an exit nozzleof the system, for example, exit nozzle 214 described above with FIG. 2.After being extracted from the sampling area of the system, fluid isrouted into cartridge housing 305 via, for example, a non-illustratedhose attached to hose part 310. Cartridge housing 305 also includesoutput aperture 322 and hose barb 325. Fluid under test passes throughoutput aperture 322 to non-illustrated blower after passing throughmicrobe collection filter, which is described in additional detailbelow.

The assembly of FIG. 3 includes cartridge 330, which includes a knurledhandle for easier handling. The function of cartridge 330 is to hold amicrobe collection filter in cartridge housing 305 such that the microbecollection filter is sealed in the flow path of fluid being extractedfrom the system. Cartridge 330 includes support plate 335. Support plate335 is, in one embodiment, a rigid disk containing perforations thatallow fluid flow. An illustrative embodiment of support plate 335 is setforth in more detail below with respect to FIG. 4. Support plate 335 issecured to cartridge 330 by a fastener 340 that engages a through-holein support plate 335.

The assembly of FIG. 3 includes microbe collection filter 345. In oneembodiment, microbe collection filter is a disk of gas-permeable, waterimpregnated gelatin, but this is not a requirement. Microbe collectionfilter need only be capable of trapping some microbes present in air towhich microbe collection filter 345 is exposed. Microbe collectionfilter is secured into cartridge 330 with o-ring 350 and clamp ring 355.Once assembled cartridge 330 is inserted into and sealed againstcartridge housing 305 with additional o-rings 360, 365. Cartridge 330 issecured in cartridge housing 305 using a plurality of pins 370. Uponassembly microbe collection filter 345 is sealed in the fluid flow pathemerging from the system such that all of the fluid emerging from thesystem is forced through microbe collection filter 345.

FIG. 4 shows an exemplary filter support plate suitable for use inconjunction with an embodiment of the invention. In one embodiment,filter support plate is approximately 47 mm in diameter. The filtersupport plate of FIG. 4 includes a central aperture 405 having adiameter of approximately 3 mm. Central aperture 405 is optionallychamfered to receive a non-illustrated fastener. Filter support plateincludes an outside annular zone 407, configured to lay outside the areaof microbe collection filter beings supported by the filter supportplate. Outside annular zone 407 is optionally used for mounting orsealing to the filter support plate. In one embodiment, the filtersupport plate of FIG. 4 is approximately 1 mm thick.

The filter support plate of FIG. 4 includes a plurality of perforations410 that allow air to flow from front to back. Although circular,radially arranged perforations are shown with respect to the filtersupport plate of FIG. 4, this not a requirement. The only requirement isthat the filter support plate includes perforations sufficient to allowair to flow through a gas-permeable microbe collection filter, e.g., agelatin wafer, in contact with the filter support plate whilemechanically supporting the microbe collection filter againstdeformations caused by air pressure.

FIG. 5 shows a method of detecting and identifying microbes according toan embodiment of the invention. In the method of FIG. 5 particles in afluid under test, for example air, are optically characterized. Opticalcharacterization is conducted by illuminating the fluid under test witha beam of light, for example a beam of light generated by a laser or LEDemitting at a wavelength of between 270 nm and 410 nm. Particles presentin the fluid scatter light by Mie scattering at various angles dependingon the size of the scattering particle. Additionally, biologicalparticles in the fluid absorb light and re-radiate light at longerwavelengths by fluorescence. Light that is scattered into predeterminedangles by particles in the fluid is detected and used to determine thesize of the scattering particles. At the same time, light emitted byfluorescence from the particles is detected and used to make adetermination as to whether the particles are biological ornon-biological. The scattered light and fluorescence measurements aretemporally correlated to determine the size of biological andnon-biological particles present in the fluid being measured.Additionally, the scattered light and fluorescence measurements arestored, for example as a function of time, for later use and analysis.

After the fluid being measured is optically characterized, that fluid isexposed to a gas-permeable microbe collection filter, e.g., a gelatinwafer. After a predetermined period of time, the gas-permeable microbecollection filter is removed and cultured to encourage microbial growth.The culturing process comprises providing nutrients to microbes on theexposed gas-permeable microbe collection filter, and incubating same.After an incubation period has elapsed, the cultured microbe collectionfilter is analyzed. Such analysis can occur by any number of means, forexample, colony counting, observing the macroscopic appearance of thegrowth patterns of microbes on the filter, microscope observation of themicrobes, chemical testing for the metabolic by-products of microbialgrowth, or DNA testing.

If the analysis applied to the incubated filter determines the number ofmicrobes that were present in the filter after the exposure period, thisnumber is, in one embodiment, correlated with the optically measureddata on the size and number of biological particles detected during theexposure period. Accordingly, correlating the optically measured datawith the data from analysis of the cultured filter can be as simple ascomparing the microbe count from the filter with the number of opticallydetected particles.

If the analysis applied to the incubated filter determines the types ofmicrobes present on the filter at the time of exposure, thisinformation, in one embodiment, is correlated with the stored data onthe size and biological status of particles measured optically at thetime the filter was exposed. This correlation optionally results in adetermination of the number and types of microbes present in the fluidsample during the measurement period, i.e., the period of time thefilter was exposed to fluid for which optical data was collected. Thisdetermination is made with the aid of conventional knowledge of the sizeof the types microbes identified on the filter by the analysis of theincubated filter.

The method illustrated in FIG. 5 has a number of advantages. First, themethod of FIG. 5 provides a more complete retrospective characterizationof the measured fluid sample by including information about the type ofmicrobes that were instantaneously detected. Second, data collectedaccording to the method of FIG. 5 allows for tentative, real-timecharacterization of future biological contamination events based onpreviously measured data. For example, suppose a contamination eventoccurs that results in numerous biological particles in the 0.4-0.8 μmrange, which are instantaneously detected by the optical systems andmethods set forth above. Upon analysis of a growth-medium containingfilter exposed to air measured during this hypothetical contaminationevent, it is determined that most of these biological particles were aparticular kind of microbe. In the future, whenever a spike ofbiological particles in the 0.4-0.8 μm range is detected under similarconditions, it can be assumed that another contamination eventassociated with the previously detected microbe is occurring in themonitored environment, and a real-time response appropriate to thatcontamination event can be coordinated. A predictive step is illustratedin FIG. 5, where microbial characteristics of a contamination event arepredicted by matching optically collected data with previously opticallycollected data that was previously correlated to a particular set ofidentified microbes.

Computer hardware, software and machine vision components can be used toperform, assist or simplify and of the process steps performed herein.FIG. 6 shows an exemplary computer system for performing analysis of afluid under test according to an embodiment of the invention. The systemof FIG. 6 includes a particle sizing, biological particleidentification, and microbial collection system 605. In certainembodiments, system 605 is the system described above with respect toFIGS. 1 and 2. System 605 performs real-time particle detection andsizing as well as real-time determination of whether detected particlesare biological or non-biological. Additionally, system 605 performsmicrobial collection by exposure of a microbe collection filter to thefluid being optically measured. The optically measured data is convertedto electrical signals by electronics included with system 605, forexample, by the drive electronics associated with a photo-diodemeasuring optical scattering and a PMT measuring fluorescence light. Theelectrical signals generated by these two detectors are transmitted fromsystem 605 to optional computer data acquisition card 610, which iselectronic communication with computer 615. Optically measured data isthen stored in a persistent storage medium, for example, hard disk 620.

The system of FIG. 6 further includes machine vision camera 630, whichis electronic communication with image capture board 625, which in turnis in electronic communication with computer 615. After a microbecollection filter 635 has been incubated, and microbial colonies arevisible, camera 630 captures an image of the colonies visible on filter635. To assist in this task, optional non-illustrated steppers may beused to translate the field of view of camera 630 with respect to filter635 or vice-versa. An image or images of the filter showing microbialcolonies is stored by computer 615 to disk 620.

Disk 620 also includes computer readable instructions 622 operable tocause computer 615 to correlate and/or compare the colony count detectedby camera 630 with the optically measured data regarding the number andbiological or non-biological status of particles measured during thetime period when filter 635 was exposed. More generally, embodiments ofthe invention include instructions, such as instructions 622, residingin computer readable medium, such as for example computer hard drive 620wherein those instructions are executed by a processor, such asprocessor residing in computer 615, to perform one or more of stepsillustrated with respect to FIG. 5, for example the storage step,analyze step, correlate step or predict step. Computer readableinstructions 622 need not reside on hard disk 620, but may reside in anymedium capable of being in electronic communication with a processorcapable of executing instructions 622. For example, there is norequirement that data (e.g., optically measured particle data) be storedon the same medium that includes instructions 622. Additionally, whileFIG. 6 shows computer 620, camera 630 and other components beingseparate from system 605, this is not a requirement. System 605 could beconfigured to include a microprocessor, storage, memory, input/outputelectronics and a camera necessary to perform the method steps describedherein.

In other embodiments, Applicants' invention includes instructionsresiding in any other computer program product, where those instructionsare executed by a computer external to, or internal to, systems such assystem 605, to perform one or more steps described with respect to FIG.5. In either case, the instructions may be encoded in computer readablemedium comprising, for example, a magnetic information storage medium,an optical information storage medium, an electronic information storagemedium, and the like. By “electronic storage media,” Applicants mean,for example and without limitation, one or more devices, such as andwithout limitation, a PROM, EPROM, EEPROM, Flash PROM, compactflash,smartmedia, and the like.

While the preferred embodiments of the present invention have beenillustrated in detail, it should be apparent that modifications andadaptations to those embodiments may occur to one skilled in the artwithout departing from the scope of the present invention as set forthin the following claims.

What is claimed is:
 1. A microbial detection and identification systemcomprising: a sampling area; a detector capable of measuring particlesin a fluid received in the sampling area; a source of pressure capableof ensuring a flow of fluid through the sampling area as said particlesare being measured by the detector; and a filter holder adapted to holda microbe collection filter having a front side, a rear side, and afluid permeable interior volume, wherein the filter holder includes: arecess for holding said microbe collection filter such that the frontside of said microbe collection filter is in fluid communication withsaid sampling area; at least one perforation in arranged to contact therear side of the microbe collection filter, and in fluid communicationwith an exhaust port.
 2. The system of claim 1, wherein the source ofpressure is a blower in fluid communication with the sampling area, andwherein the blower is arranged between and in fluid communication withthe at least one perforation of the filter holder and the exhaust port.3. The system of claim 1, wherein said filter holder is configured tohold the microbe collection filter such that its front side faces thesampling area.
 4. The system of claim 1, wherein said detector comprisesa sizing detector capable of sizing particles in the fluid.
 5. Thesystem of claim 1, wherein said detector comprises a viability detectorcapable of determining viability of particles in the fluid.
 6. Thesystem of claim 1, wherein said detector includes a light source thatilluminates particles to be measured in the sampling area.
 7. Theparticle detector of claim 6, wherein the detector includes both asizing detector capable of sizing particles in the fluid and a viabilitydetector capable of determining viability of particles in the fluid. 8.The system of claim 7, wherein the sizing detector sizes particles onthe basis of detecting light scattered into a predetermined angularrange by particles of a predetermined size, and wherein said viabilitydetector determines viability by measuring light emitted by fluorescencefrom illuminated biological particles in said sampling area.
 9. Thesystem of claim 1, wherein the recess is substantially circular.
 10. Thesystem of claim 1, wherein the at least one perforation comprises aplurality of perforations arranged in a radial pattern.
 11. The systemof claim 1 further comprising a microbe collection filter.
 12. Thesystem of claim 11, wherein said microbe collection filter comprises amedium capable of adhering particles contained in a fluid that passthrough the medium.
 13. The system of claim 12, wherein the medium iscapable of sustaining microbial viability for particles adhered to themedium for a time sufficient to allow the filter to be removed toanalyze the particles.
 14. The system of claim 13, wherein the medium isgelatin.
 15. The system of claim 13, wherein the medium includes amoisture content sufficient to sustain microbial viability for particlesadhered to the medium for a time sufficient to allow the filter to beremoved to analyze the particles.
 16. A microbial detection andidentification system comprising: a sampling area; a detector capable ofmeasuring particles present in a fluid received in the sampling area; asource of pressure capable of ensuring flow of fluid through thesampling area as said particles are being measured by the detector; aremovable filter holder capable of holding a removable microbecollection filter, the filter having a front side, a rear side, and afluid permeable interior volume, wherein the filter holder is arrangedto hold said microbe collection filter such that the front side of saidmicrobe collection filter is transverse to the flow of fluid and is influid communication with said sampling area, and further includes atleast one perforation arranged to be in contact with the rear side ofthe microbe collection filter and in fluid communication with an exhaustport.
 17. The system of claim 16, wherein the source of pressure is ablower in fluid communication with the sampling area, and wherein theblower is arranged between and in fluid communication with the at leastone perforation of the filter holder and the exhaust port.
 18. Thesystem of claim 16, wherein said detector comprises a sizing detectorcapable of sizing particles in the fluid.
 19. The system of claim 16,wherein said detector comprises a viability detector capable ofdetermining viability of particles in the fluid.
 20. The system of claim16, wherein said detector includes a light source that illuminatesparticles to be measured in the sampling area.
 21. The particle detectorof claim 20, wherein the detector includes both a sizing detectorcapable of sizing particles in the fluid and a viability detectorcapable of determining viability of particles in the fluid.
 22. Thesystem of claim 21, wherein the sizing detector sizes particles on thebasis of detecting light scattered into a predetermined angular range byparticles of a predetermined size, and wherein said viability detectordetermines viability by measuring light emitted by fluorescence fromilluminated biological particles in said sampling area.
 23. The systemof claim 16, wherein said at least one perforation comprises a pluralityof perforations arranged in radial pattern.
 24. The system of claim 16,wherein the filter holder includes a recess for receiving the microbecollection filter.
 25. The system of claim 24, wherein the recess issubstantially circular.
 26. A microbial detection and identificationsystem comprising: a sampling area for receiving a fluid; a detectorcapable of measuring particles in the received fluid; a microbecollection filter in fluid communication with said sampling area,wherein the microbe collection filter has a front side facing thesampling area, and a rear side in fluid communication with a source ofnegative pressure that supplies sufficient negative pressure on the rearside of the microbe collection filter to evacuate the fluid from saidsampling area as the particles are being measured.
 27. The system ofclaim 26, wherein when said fluid is exposed to said microbe collectionfilter, particles within said fluid adhere to said microbe collectionfilter.
 28. The system of claim 26, wherein the source of negativepressure is a blower in fluid communication with the sampling area. 29.The system of claim 26, wherein said detector comprises a sizingdetector capable of sizing particles in the fluid.
 30. The system ofclaim 26, wherein said detector comprises a viability detector capableof determining viability of particles in the fluid.
 31. The system ofclaim 26, wherein said detector includes a light source that illuminatesparticles to be measured in the sampling area.
 32. The particle detectorof claim 31, wherein the detector includes both a sizing detectorcapable of sizing particles in the fluid and a viability detectorcapable of determining viability of particles in the fluid.
 33. Thesystem of claim 32, wherein the sizing detector sizes particles on thebasis of detecting light scattered into a predetermined angular range byparticles of a predetermined size, and wherein said viability detectordetermines viability by measuring light emitted by fluorescence fromilluminated biological particles in said sampling area.
 34. The systemof claim 26, wherein the microbe collection filter is substantiallycircular.
 35. The system of claim 26, wherein said microbe collectionfilter comprises a medium capable of adhering particles contained in afluid that pass through the medium.
 36. The system of claim 35, whereinthe medium is capable of sustaining microbial viability for particlesadhered to the medium for a time sufficient to allow the filter to beremoved to analyze the particles.
 37. The system of claim 36, whereinthe medium is gelatin.
 38. The system of claim 36, wherein the mediumincludes a moisture content sufficient to sustain microbial viabilityfor particles adhered to the medium for a time sufficient to allow thefilter to removed to analyze the particles.